The present invention relates to compositions, which can be used for purifying and crystallizing molecules of interest.
Proteins and other macromolecules are increasingly used in research, diagnostics and therapeutics. Proteins are typically produced by recombinant techniques on a large scale with purification constituting the major cost (up to 60% of the total cost) of the production processes. Thus, large-scale use of recombinant protein products is hindered because of the high cost associated with purification.
Current protein purification methods are dependent on the use of a combination of various chromatography techniques. These techniques separate mixtures of proteins on the basis of their charge, degree of hydrophobicity or size among other characteristics. Several different chromatography resins are available for use with each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein targeted for isolation. The essence of each of these separation methods is that proteins can be caused either to move at different rates down a long column, achieving a physical separation that increases as they pass further down the column, or to adhere selectively to the separation medium, enabling differential elution by different solvents. In some cases, the column is designed such that impurities bind thereto while the desired protein is found in the “flow-through.”
Affinity precipitation (AP) is the most effective and advanced approach for protein precipitation [Mattiasson (1998); Hilbrig and Freitag (2003) J Chromatogr B Analyt Technol Biomed Life Sci. 790(1-2):79-90]. Current state of the art AP employs ligand coupled “smart polymers”. “Smart polymers” [or stimuli-responsive “intelligent” polymers or Affinity Macro Ligands (AML)] are polymers that respond with large property changes to small physical or chemical stimuli, such as changes in pH, temperature, radiation and the like. These polymers can take many forms; they may be dissolved in an aqueous solution, adsorbed or grafted on aqueous-solid interfaces, or cross-linked to form hydrogels [Hoffman J Controlled Release (1987) 6:297-305; Hoffman Intelligent polymers. In: Park K, ed. Controlled drug delivery. Washington: ACS Publications, (1997) 485-98; Hoffman Intelligent polymers in medicine and biotechnology. Artif Organs (1995) 19:458-467]. Typically, when the polymer's critical response is stimulated, the smart polymer in solution will show a sudden onset of turbidity as it phase-separates; the surface-adsorbed or grafted smart polymer will collapse, converting the interface from hydrophilic to hydrophobic; and the smart polymer (cross-linked in the form of a hydrogel) will exhibit a sharp collapse and release much of its swelling solution. These phenomena are reversed when the stimulus is reversed, although the rate of reversion often is slower when the polymer has to redissolve or the gel has to re-swell in aqueous medium.
“Smart” polymers may be physically mixed with, or chemically conjugated to, biomolecules to yield a large family of polymer-biomolecule systems that can respond to biological as well as to physical and chemical stimuli. Biomolecules that may be polymer-conjugated include proteins and oligopeptides, sugars and polysaccharides, single- and double-stranded oligonucleotides and DNA plasmids, simple lipids and phospholipids, and a wide spectrum of recognition ligands and synthetic drug molecules.
A number of structural parameters control the ability of smart polymers to specifically precipitate proteins of interest; smart polymers should contain reactive groups for ligand coupling; not interact strongly with the impurities; make the ligand available for interaction with the target protein; give complete phase separation of the polymer upon a change of medium property; form compact precipitates; exclude trapping of impurities into the gel structure and be easily solubilized after the precipitate is formed.
Although many different natural as well as synthetic polymers have been utilized in AP [Mattiasson (1998) J. Mol. Recognit. 11:211] the ideal smart polymers remain elusive, as affinity precipitations performed with currently available smart polymers, fail to meet one or several of the above-described requirements [Hlibrig and Freitag (2003), supra].
The availability of efficient and simple protein purification techniques may also be useful in protein crystallization, in which protein purity extensively affects crystal growth. The conformational structure of proteins is a key to understanding their biological functions and to ultimately designing new drug therapies. The conformational structures of proteins are conventionally determined by x-ray diffraction from their crystals. Unfortunately, growing protein crystals of sufficient high quality is very difficult in most cases, and such difficulty is the main limiting factor in the scientific determination and identification of the structures of protein samples. Prior art methods for growing protein crystals from super-saturated solutions are tedious and time-consuming, and less than two percent of the over 100,000 different proteins have been grown as crystals suitable for x-ray diffraction studies.
Membrane proteins present the most challenging group of proteins for crystallization. The number of 3D structures available for membrane proteins is still around 20 while the number of membrane proteins is expected to constitute a third of the proteome. Numerous obstacles need to be traversed when wishing to crystallize a membrane protein. These include, low abundance of proteins from natural sources, the need to solubilize hydrophobic membrane proteins from their native environment (i.e., the lipid bilayer) and their tendency to denaturate, aggregate and/or degrade in the detergent solution. The choice of the solubilizing detergent presents another problem as some detergents may interfere with binding of a stabilizing partner to the target protein.
Two approaches have been attempted in the crystallization of membrane proteins.
Until very recently, the majority of X-ray crystal structures of membrane proteins have been determined using crystals grown directly from solutions of protein-detergent complexes. Crystal growth of protein-detergent complexes can be considered equivalent to that of soluble proteins only the solute being crystallized is a complex of protein and detergent, rather than solely protein. The actual lattice contacts are formed by protein-protein interactions, although crystal packing brings the detergent moieties into close apposition as well. In order to increase the surface area available to make these protein-protein contacts studies suggested adding an antibody fragment which will increase the chances of producing crystals [Hunte and Michel (2002) Curr. Opin. Struct. Biol. 12:503-508]. However, applying this technology to various membrane proteins is difficult as it requires the generation of monoclonal antibodies, which are specific to each membrane protein.
Furthermore, it is argued that no detergent micelle can fully and accurately reproduce the lipid bilayer environment of the protein.
Thus, efforts to crystallize membrane proteins must be directed towards producing crystals within a bilayer environment. A number of attempts have been made to generate crystals of membrane proteins using this approach. These include the generation of crystals of bacteriorhodopsin grown in the presence of a lipidic cubic phase, which forms gel-like substance containing continuous bilayer structures [Landau and Rosenbuch (1996) Proc. Natl. Acad. Sci. USA 93:14532-14535] and crystallization in cubo which was proven successful in the crystallization of archaeal seven-transmembrane helix proteins [Gordeliy (2002) Nature 419:484-487; Luecke (2001) Science 293:1499-1503; Kolbe (2000) Science 288:1390-1396; Royant (2001) Proc. Natl. Acad. Sci. USA 98:10131-10136]. However, crystals of other membrane proteins using the in cubo approach were not of as high a quality as crystals grown directly from protein-detergent complex solutions [Chiu (2000) Acta. Crystallogr. D. 56:781-784].
There is thus a widely recognized need for, and it would be highly advantageous to have, compositions and methods using same for the purification and crystallization of molecules which are devoid of the above limitations.